Filtration of Gene Trees From 9,000 Exons, Introns, and UCEs Disentangles Conflicting Phylogenomic Relationships in Tree Frogs (Hylidae)

An emerging challenge in interpreting phylogenomic data sets is that concatenation and multi-species coalescent summary species tree approaches may produce conflicting results. Concatenation is problematic because it can strongly support an incorrect topology when incomplete lineage sorting (ILS) results in elevated gene-tree discordance. Conversely, summary species tree methods account for ILS to recover the correct topology, but these methods do not account for erroneous gene trees (“EGTs”) resulting from gene tree estimation error (GTEE). Third, site-based and full-likelihood methods promise to alleviate GTEE as these methods use the sequence data from alignments. To understand the impact of GTEE on species tree estimation in Hylidae tree frogs, we use an expansive data set of ∼9,000 exons, introns, and ultra-conserved elements and initially found conflict between all three types of analytical methods. We filtered EGTs using alignment metrics that could lead to GTEE (length, parsimony-informative sites, and missing data) and found that removing shorter, less informative alignments reconciled the conflict between concatenation and summary species tree methods with increased gene concordance, with the filtered topologies matching expected results from past studies. Contrarily, site-based and full-likelihood methods were mixed where one method was consistent with past studies and the other varied markedly. Critical to other studies, these results suggest a widespread conflation of ILS and GTEE, where EGTs rather than ILS are driving discordance. Finally, we apply these recommendations to an R package named PhyloConfigR, which facilitates phylogenetic software setup, summarizes alignments, and provides tools for filtering alignments and gene trees

Resistome Identification from Whole Genome Sequencing Data of Norwegian Isolates

Masters in Applied and Commercial Biotechnology. Inland Norway University of Applied Sciences. Faculty of Applied Ecology, Agricultural sciences and BiotechnologyAntimicrobial resistance (AMR) is considered a potential threat to global health. Norway have had a low prevalence of resistant bacteria. But in the recent years there has been an increase in resistant bacteria including, Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii. Traditionally, clinical microbiology has used culture-based techniques to determine antimicrobial susceptibility and resistance profiles, but now whole–genome sequencing for antibiotic susceptibility (WGS-AST) has emerged as a potential alternative. We aimed to investigate the prevalence of antimicrobial resistance genes and plasmids in WGS of 111 clinical Norwegian isolates of E. coli, K. pneumoniae, and A. baumannii, to identify correlations between phenotypic and genotypic resistance in the isolates, which are related to antibiotic resistance to β-lactam, aminoglycosides, fluoroquinolone, trimethoprim, tetracycline, and phenicol. The most occurring drug class was β-lactam antibiotic with TEM (38%) in E.coli, SHV (67%) in K. pneumoniae, and OXA (100%) and TEM (45%) gene families in A. baumannii. In silico detection of plasmids with Brooks et al database showed plasmid p2_000837 as prominent plasmid 12% E.coli isolates. There were four plasmids (pIB_NDM_1, p2_W5-6, pCHL5009T-102k-mcr3, pVir_020022) in 2% K. pneumoniae isolates which were also shared with E. coli. Only one plasmid (pHZ23-1-1) was confirmed in 9% of A. baumannii isolates. PLSDB detected Plasmid A and plasmid 4 with the maximum percentage in E.coli (10%) and K. pneumoniae isolates (4%). In E. coli and K. pneumoniae, the presence of incompatibility groups was observed; IncFIB (64% and 27%), Col156 (74% and 27%), IncFII (43% and 15%), while IncHI-1B(pNDM-MAR) (12%) were present only in K. pneumoniae . A total of 75 isolates had resistance to the tested β-lactam antibiotics, out of which 63 had the corresponding resistance genes (ampC, SHV, CTX-M, TEM, LEN, OXA). Only 11 E.coli and one K. pneumoniae isolates were found to have resistance genes and the plasmids on the same node to confirm plasmid mediated resistance. This study demonstrates the utility of WGS in defining resistance elements and highlights the diversity of resistance within the selected isolates to further the diagnostics and therapeutics for the treatment of the relevant infections

Building a model: developing genomic resources for common milkweed (Asclepias syriaca) with low coverage genome sequencing

<p>Abstract</p> <p>Background</p> <p>Milkweeds (<it>Asclepias </it>L.) have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (<it>Asclepias syriaca </it>L.) could be useful in characterizing the genome of a plant without prior genomic information and for development of genomic resources as a step toward further developing <it>A. syriaca </it>as a model in ecology and evolution.</p> <p>Results</p> <p>A 0.5× genome of <it>A. syriaca </it>was produced using Illumina sequencing. A virtually complete chloroplast genome of 158,598 bp was assembled, revealing few repeats and loss of three genes: <it>accD, clpP</it>, and <it>ycf1</it>. A nearly complete rDNA cistron (18S-5.8S-26S; 7,541 bp) and 5S rDNA (120 bp) sequence were obtained. Assessment of polymorphism revealed that the rDNA cistron and 5S rDNA had 0.3% and 26.7% polymorphic sites, respectively. A partial mitochondrial genome sequence (130,764 bp), with identical gene content to tobacco, was also assembled. An initial characterization of repeat content indicated that Ty1/<it>copia</it>-like retroelements are the most common repeat type in the milkweed genome. At least one <it>A. syriaca </it>microread hit 88% of <it>Catharanthus roseus </it>(Apocynaceae) unigenes (median coverage of 0.29×) and 66% of single copy orthologs (COSII) in asterids (median coverage of 0.14×). From this partial characterization of the <it>A. syriaca </it>genome, markers for population genetics (microsatellites) and phylogenetics (low-copy nuclear genes) studies were developed.</p> <p>Conclusions</p> <p>The results highlight the promise of next generation sequencing for development of genomic resources for any organism. Low coverage genome sequencing allows characterization of the high copy fraction of the genome and exploration of the low copy fraction of the genome, which facilitate the development of molecular tools for further study of a target species and its relatives. This study represents a first step in the development of a community resource for further study of plant-insect co-evolution, anti-herbivore defense, floral developmental genetics, reproductive biology, chemical evolution, population genetics, and comparative genomics using milkweeds, and <it>A. syriaca </it>in particular, as ecological and evolutionary models.</p

Overcoming tumor resistance by heterologous adeno-poxvirus combination therapy

Successful cancer control relies on overcoming resistance to cell death and on activation of host antitumor immunity. Oncolytic viruses are particularly attractive in this regard, as they lyse infected tumor cells and trigger robust immune responses during the infection. However, repeated injections of the same virus promote antiviral rather than antitumor immunity and tumors may mount innate antiviral defenses to restrict oncolytic virus replication. In this article, we have explored if alternating the therapy virus could circumvent these problems. We demonstrate in two virus-resistant animal models a substantial delay in antiviral immune- and innate cellular response induction by alternating injections of two immunologically distinct oncolytic viruses, adenovirus, and vaccinia virus. Our results are in support of clinical development of heterologous adeno-/vaccinia virus therapy of cancer.Peer reviewe

Towards fidelity, integrity and authenticity: A critical reflection of an academic-oriented mindfulness-based programme

Despite a burgeoning evidence-base (Black 2018), there exists a number of issues and debates in the developing field of mindfulness that impact upon its pedagogy. These create an “unstable ground” (McCown, Reibel and Micozzi, 2010, p3) for teachers of mindfulness, such as myself. According to McCown (2017; 2014), tensions often arise from the disconnects between drives to create a standardised, structured account of mindfulness for teaching, training and research (fidelity) and efforts to authentically express the constructivist nature of the pedagogy of mindfulness in its teaching (integrity). In this study I utilised a critical realist approach to evaluate the outcomeeffectiveness and pedagogical properties of an established and popular Academic- Oriented Mindfulness Programme (AO-MBP) course offered to adult learners at a leading UK university. Heeding the calls in the field to develop new methodological approaches (e.g. Grossman, 2011; Brown et al., 2011), I employed a ‘convergent parallel phase’ mixed-methods design that analysed data collected from multiple sources. The analytical process of retroduction produced four overarching tendencies for the course. These were 1) that the pedagogic approach was effective in balancing the demands of the course; 2) that the learners of the course experienced significant growth in mindfulness and associated benefits; 3) that the course was effective as an HE-based academic-oriented course, and; 4) that there were possible pedagogic developments that would move the course towards greater fidelity, integrity and authenticity. Causal mechanisms that underpinned the expression of these tendencies were theorised and included factors concerning the teacher, the course, the learners and the wider field of mindfulness. A guidance framework for the design, development, implementation and evaluation of AO-MBPs was produced from the findings. Applications are discussed alongside the wider implications for the cultivating of genuinely transformational mindfulness-based programmes

Reinventing residual reserves in the sea: Are we favouring ease of establishment over need for protection?

© 2014 The Authors. As systems of marine protected areas (MPAs) expand globally, there is a risk that new MPAs will be biased toward places that are remote or unpromising for extractive activities, and hence follow the trend of terrestrial protected areas in being 'residual' to commercial uses. Such locations typically provide little protection to the species and ecosystems that are most exposed to threatening processes. There are strong political motivations to establish residual reserves that minimize costs and conflicts with users of natural resources. These motivations will likely remain in place as long as success continues to be measured in terms of area (km2) protected. The global pattern of MPAs was reviewed and appears to be residual, supported by a rapid growth of large, remote MPAs. The extent to which MPAs in Australia are residual nationally and also regionally within the Great Barrier Reef (GBR) Marine Park was also examined. Nationally, the recently announced Australian Commonwealth marine reserves were found to be strongly residual, making almost no difference to 'business as usual' for most ocean uses. Underlying this result was the imperative to minimize costs, but without the spatial constraints of explicit quantitative objectives for representing bioregions or the range of ecological features in highly protected zones. In contrast, the 2004 rezoning of the GBR was exemplary, and the potential for residual protection was limited by applying a systematic set of planning principles, such as representing a minimum percentage of finely subdivided bioregions. Nonetheless, even at this scale, protection was uneven between bioregions. Within-bioregion heterogeneity might have led to no-take zones being established in areas unsuitable for trawling with a risk that species assemblages differ between areas protected and areas left available for trawling. A simple four-step framework of questions for planners and policy makers is proposed to help reverse the emerging residual tendency of MPAs and maximize their effectiveness for conservation. This involves checks on the least-cost approach to establishing MPAs in order to avoid perverse outcomes

BIOCHEMICAL METHODS FOR PREPARATION AND STUDY OF PEPTIDE NATURAL PRODUCT LIBRARIES

mRNA Display is an increasingly popular technique in pharmaceutical sciences to make highly diverse peptide libraries to pan for protein inhibitors. The current state of the art applies Flexizyme codon reprogramming with mRNA display to introduce unnatural amino acids for peptide cyclization and to further increase library diversity. Interestingly, ribosomally synthesized and post-translationally modified peptides (RiPP) are a unique class of natural products that transform linear peptides into highly modified and structurally complex metabolites. By combining RiPP biosynthesis with mRNA display, libraries of increasingly greater diversity can be achieved, and impending selected inhibitors will have natural product-like qualities, which we expect will allow these compounds to have better drug-like properties. Herein, we have developed a platform to measure RiPP enzyme modification of mRNA display libraries to show for the first time that RiPP enzymes can modify RNA linked peptide substrates. The platform may be extrapolated to many different RiPP enzymes and provides useful measurements to determine if a RiPP enzyme is promiscuous and effective to produce highly diversified peptides. Thiopeptides are a specific class of RiPP that we would like to apply to mRNA display, because they have broad activities. Additionally, the structure of thiopeptides is primed for mRNA display protocols, consisting of a highly decorated macrocycle with a C-terminal tail that may readily accept an mRNA tag. We present a redesigned chemoenzymatic strategy to make thiopeptides using cell free protein synthesis that is promiscuous, can synthesize different thiopeptide classes, and is mRNA display ready. Finally, bioinformatic analysis of publicly available thiopeptide gene clusters shows that many unknown thiopeptides still exist. We have organized a set of 14 new clusters for which we can apply our chemoenzymatic strategy and characterize these unknown compounds, with the hope of finding the best thiopeptide enzymes for use in mRNA display selections.Doctor of Philosoph

Landscape drivers of genomic diversity and divergence in woodland Eucalyptus

Spatial genetic patterns are influenced by numerous factors, and they can vary even among coexisting, closely related species due to differences in dispersal and selection. Eucalyptus (L'Héritier 1789; the “eucalypts”) are foundation tree species that provide essential habitat and modulate ecosystem services throughout Australia. Here we present a study of landscape genomic variation in two woodland eucalypt species, using whole-genome sequencing of 388 individuals of Eucalyptus albens and Eucalyptus sideroxylon. We found exceptionally high genetic diversity (π ≈ 0.05) and low genome-wide, interspecific differentiation (FST = 0.15) and intraspecific differentiation between localities (FST ≈ 0.01–0.02). We found no support for strong, discrete population structure, but found substantial support for isolation by geographic distance (IBD) in both species. Using generalized dissimilarity modelling, we identified additional isolation by environment (IBE). Eucalyptus albens showed moderate IBD, and environmental variables have a small but significant amount of additional predictive power (i.e. IBE). Eucalyptus sideroxylon showed much stronger IBD and moderate IBE. These results highlight the vast adaptive potential of these species and set the stage for testing evolutionary hypotheses of interspecific adaptive differentiation across environmentsAustralian Research Council, Grant/Award Number: CE140100008, DP150103591 and DE19010032